For many years, bronchial asthma was regarded as an abnormality of respiratory smooth muscle in which afflicted individuals experience the onset of bronchospasm as a consequence of overreactivity of the bronchial smooth muscle. Later, the bronchial mast cell was thought to play a critical role in the stimulation of bronchial smooth muscle by producing leukotriene C4 (the slow-reacting substance of anaphylaxis) and histamine, which cause contraction. However, over the past few years, a dramatic change in thinking regarding the pathophysiology of bronchial asthma has occurred and the involvement of eosinophilic leukocytes, or "eosinophils," in the inflammation of the airway has been suspected.
Eosinophils are a type of leukocyte containing cytoplasmic granules that stain strongly with acidic dyes. Eosinophils have been associated with bronchial asthma since the early part of this century and they are characteristically found in large numbers in the lung tissue of patients dying of asthma (A. G. Ellis et al., J. Med. Sci., 136, 407 (1908)). In the mid 1970s, it was demonstrated that the severity of bronchial asthma can be related to the number of eosinophils in the peripheral blood of the patients (B. R. Horn et al., N. Engl. J. Med., 292, 1152 (1975)). Also around this time, studies of eosinophils had shown the presence of basic (cationic) granule proteins. One of the principal proteins associated with eosinophil granules, the major basic protein (MBP), was so-named because, in the guinea pig, it comprises more than 50% of the granule protein, is strongly basic (arginine-rich), and is proteinaceous (G. J. Gleich, J. Exp. Med., 137, 1459 (1973); T. L. Wasmoen et al., J. Biol. Chem., 263, 12559 (1988)). MBP is toxic to worms (helminths) and mammalian cells, and causes damage to bronchial respiratory epithelium (G. J. Gleich et al., Adv. Immunol., 39, 177 (1986)). For example, direct application of MBP to respiratory epithelium in concentrations as low as 10 .mu.g/ml (7.1.times.10.sup.-7 M) causes ciliostasis and epithelial damage. This damage consists of desquamation of epithelial cells into the lumen of the respiratory tract, as well as frank disruption of epithelial cells. The effects of MBP are dose-related and higher doses cause damage more quickly and to a greater extent than lower doses (E. Frigas et al., Lab. Invest., 42, 35 (1980)). These effects are caused both by MBP from guinea pig eosinophils and from human eosinophils, and impact both guinea pig and human respiratory tissues (G. J. Gleich et al., J. Immunol., 123, 2925 (1979)).
The findings that MBP causes ciliostasis, desquamation of respiratory epithelial cells, and damage to the respiratory epithelial cells are suggestive of the pathologic changes observed in bronchial asthma. In bronchial asthma, an exudate of eosinophils, normal and degenerating bronchial epithelial cells, and clumps of epithelial cells, referred to as Creola bodies, are present in the bronchial lumen. In the bronchial mucosa and submucosa, edema, separation and shedding of ciliated cells, and eosinophil infiltration are seen. Thus, the effects of the eosinophil granule MBP in vitro are similar to the pathology characteristic of bronchial asthma (M. S. Dunnill, J. Clin. Path., 13, 27 (1960)).
Because of this discovery, the levels of MBP in sputum of patients with bronchial asthma were measured to determine whether they were elevated and to what degree. Levels of MBP in sputum samples from 206 patients with various respiratory diseases were measured by radioimmunoassay. In 165 of these patients, MBP was not measurable or the concentrations of MBP were less than 0.1 .mu.g/ml. In these 165 patients, only one patient carried the diagnosis of asthma. Among 41 patients with sputum concentrations of MBP greater than 0.1 .mu.g/ml, 28 were diagnosed as having asthma and in the remaining 13 patients, six had obstructive lung disease which is often confused with asthma. In 15 patients hospitalized for treatment of asthma, sputum MBP levels ranged from 0.5 (0.04.times.10.sup.-6 M) to 93 .mu.g/ml (6.6.times.10.sup.-6 M) (geometric mean 7.1 .mu.g/ml, 0.51.times.10.sup.-6 M). Further, the levels of sputum MBP in these 15 patients declined during therapy with glucocorticoids (E. Frigas et al., Mayo Clinic. Proc., 56, 345 (1981)). These results indicated that MBP levels in the toxic range were present in the sputum of patients with asthma, that levels of sputum MBP were highest in acutely ill patients, and that sputum MBP levels decline after steroid therapy.
The possibility that MBP directly causes damage to bronchial epithelium was tested utilizing immunofluorescence localization of MBP in lung tissues of patients dying of asthma (W. Filley et al., Lancet, 2, 11 (1982)). These results showed that the patients dying of asthma had the classical pathologic features of bronchial asthma with a thickened basement membrane zone, goblet cell hyperplasia, and peribronchial inflammatory infiltrates with eosinophils in the lamina propria. Examination of these same sections by immunofluorescence to localize MBP, revealed MBP deposition onto damaged bronchial epithelium. These results demonstrate that MBP was released from the eosinophil and was present in tissues at the site of damage.
Subsequent studies extended these observations showing that not only MBP, but two of the other cationic eosinophil granule proteins, namely eosinophil peroxidase (EPO) and eosinophil cationic protein (ECP), have the capacity to damage bronchial epithelium (S. Motojima et al., Am. Rev. Respir. Dis., 139, 801 (1989)). Analyses of the effect of MBP on respiratory epithelium showed that although MBP reduced the frequency of ciliary beating, its predominant effect was to reduce the number of beating ciliated cells. The effect of MBP in causing cessation of ciliary beating was seen in respiratory epithelial cells in the epithelium itself as well as in axonemes (the contractile elements of the cilia) (A. T. Hastie et al., Am. Rev. Resp. Dis., 135, 845 (1987)).
One of the signal abnormalities in bronchial asthma is bronchial hyperreactivity. Bronchial hyperreactivity is manifested in patients as a marked irritability of the respiratory tract to nonspecific stimuli including cold air, dust, and, in the laboratory, to inhaled methacholine. Indeed, this hyperreactivity is a diagnostic criterion for asthma (N. J. Gross et al., in Allergy, Principles and Practice, Vol. I., E. Middleton, Jr. et al., eds. (1988) at page 790). Analyses of MBP in the lung secretions of patients with asthma (obtained by lavage of the bronchi and alveoli) showed that MBP levels in lung fluids are correlated with bronchial hyperreactivity (A. J. Wardlaw et al., Am. Rev. Resp. Dis., 137, 62 (1988)). In cynomolgus monkeys, provocation of inflammation rich in eosinophils was associated with an increase in bronchial hyperreactivity and with the presence of MBP in lung secretions; both the numbers of eosinophils and the MBP concentration were significantly correlated with bronchial hyperreactivity to methacholine (R. H. Gundel et al., J. Appl. Physiol., 68 779 (1990)).
At the molecular level, eosinophil proliferation and differentiation are regulated by various cytokines, such as IL-3, IL-5 and GM-CSF. See D. S. Silberstein et al., Hematol. Oncol. Clin. North Am., 3, 511 (1989). These cytokines, as well as IFN-.gamma., have been shown to prolong survival of eosinophils in vitro by T. Valerius et al., J. Immunol., 145, 2950 (1990), and to augment eosinophil function (M. E. Rothenberg et al., J. Clin. Invest., 81 1986 (1988); T. Fujisawa et al., J. Immunol., 144, 642 (1990); D. S. Silberstein et al., J. Immunol., 137, 3290 (1986)). Furthermore, IL-5 primes eosinophils for enhanced locomotor responses to chemotactic agents, such as platelet-activating-factor, leukotriene B4, and IL-8 (R. Sehmi et al., Blood, 79, 2952 (1992)). Also, recent information indicates that IL-5 is present in the lung following allergen-induced pulmonary late allergic reactions (J. B. Sedgwick et al., Am. Rev. Respir. Dis., 144, 1274 (1991) and mRNA for IL-5 is expressed in the bronchial epithelium of patients with asthma (Q. Hamid et al., J. Clin. Invest., 87, 1541 (1991)). These observations suggest that the inflammation associated with asthma is critically dependent on the presence of cytokines, especially IL-5, and recent data showing that antibodies to IL-5 block both antigen-induced eosinophilia and antigen-induced bronchial hyperreactivity support that view (P. J. Mauser et al., Am. Rev. Respir. Dis., 145, A859 (1992)).
Glucocorticoids are the most useful class of drugs for treating many eosinophil-related disorders, including bronchial asthma (R. P. Schleimer et al., Am. Rev. Respir. Dis., 141, 559 (1990)). They produce eosinopenia in normal persons, decrease circulating eosinophils in patients with eosinophilia, and reduce eosinophil influx at inflammatory sites (J. H. Butterfield et al., in Antiinflammatory Steroid Action: Basic and Clinical Aspects, R. P. Schleimer et al., eds., Academic Press, Inc. (1989) at p. 151. The mechanism of these effects is still uncertain. Lamas et al. in J. Immunol., 142, 3978 (1989) and J. Allergy Clin. Immunol., 85, 282 (1990) have reported that supernatants from human vascular endothelial cells cultured with glucocorticoids had reduced eosinophil survival-enhancing activity in vitro.
Recently, N. Wallen et al., J. Immunol., 147, 3940 (1991) reported the dose-dependent inhibition of IL-5-mediated eosinophil survival by dexamethasone, methylprednisolone and hydrocortisone, and the inhibition of IL-3-, GM-CSF-, and IFN-.gamma.-mediated eosinophil survival by dexamethasone. Dexamethasone produced a dose-dependent increase in the EC.sub.50 for IL-5-mediated viability enhancement. The relative eosinophil viability inhibitory potencies of the glucocorticoids tested correlated with previously described antiinflammatory potencies and with the affinities for the glucocorticoid receptor: dexamethasone&gt;methylprednisolone&gt;hydrocortisone.
However, for many patients with asthma, glucocorticoids are the principal therapy and these patients may require glucocorticoid therapy for long periods of time, e.g., months to years. In fact, the disease can be characterized as one of chronic glucocorticoid toxicity, in that the toxicity of these steroids can cause severe morbidity and even mortality in the patients. Furthermore, cessation of glucocorticoid therapy leads to withdrawal symptoms, such as malaise and muscle pain. However, presently glucocorticoids are the only effective therapy for severe asthma, and are prescribed long-term despite their toxicity.
The information discussed above pertains to bronchial asthma and the role of toxic eosinophil granule proteins exemplified by MBP in the pathophysiology of bronchial asthma. Evidence exists that these toxic proteins also contribute to the pathogenesis of diseases associated with eosinophil infiltration in the upper respiratory tract. For example, G. H. Ayars et al. in Am. Rev. Resp. Dis., 140, 125 (1989), have reported that MBP is toxic to respiratory epithelium from the nose, and R. Bascom et al., in J. Allergy Clin. Immunol., 84, 338 (1989) found that elevated MBP concentrations are present in nasal fluids following experimental hay fever. As reported by S. L. Harlin et al., J. Allergy Clin. Immunol., 81, 867 (1988), MBP is deposited on respiratory epithelium of the upper airway in association with damage to the epithelium. Therefore, toxic eosinophil granule proteins may cause disease of the upper airway in the same manner as they likely do in the lower airway in the case of bronchial asthma.
Finally, I. J. Udell et al., in Am. J. Ophthamol., 92, 824 (1981) reported that MBP is elevated in tears of patients with vernal conjunctivitis, a form of allergic inflammation of the eye, and S. D. Trocme et al., in Am. J. Ophthamol., 108, 57 (1989) found that MBP is deposited into inflamed conjunctiva of such patients. Thus, evidence exists that MBP may act as a toxin to the conjunctiva.
Therefore, a need exists for improved therapeutic methods to treat hypersensitivity diseases, such as bronchial asthma, which are caused by, or aggravated by, eosinophils or the toxic proteins released by eosinophils.